1. Field of the Invention
The present invention relates to a crystallizing method for producing a biopolymer crystal, which is typically represented by a protein, and a crystallizing apparatus for use in implementing the method.
2. Background Art
Making clear a three-dimensional structure of a biopolymer, which is typically represented by a protein, not only enables to elucidate physiological functions in vivo of the biopolymer, but also is extremely useful from the viewpoint of achieving rational development of medicines (drug design). As a practical method for analyzing a three-dimensional structure of the biopolymer, an NMR (nuclear magnetic resonance) method and an X-ray crystal structure analysis method have been heretofore known. From the standpoint of analysis, the X-ray crystal structure analysis method in which there is no restriction in molecular weight of biopolymer is considered to be especially effective in the future. In this X-ray crystal structure analysis method, however, it is essential to prepare a single biopolymer crystal for conducting the required analysis. Moreover, to improve a resolution in the structure analysis, it is required to form a single crystal having a good crystallinity.
As a method for preparing a single biopolymer crystal, for example, a protein, a vapor diffusion method is commonly employed these days. In a sitting drop vapor diffusion method, as shown in FIG. 14, an aqueous solution 1 containing a small amount of protein of about 1 μl is dropped into a concave part 3 within a well solution retention plate 2 (in a hanging drop vapor diffusion method, an aqueous solution 1 containing a small amount of protein is dropped onto a surface being a lower face side of a cover glass 6 and adhered thereto). Further, a precipitant 5 is received in an inner bottom portion of a container-shaped concave part (well) 4, and a top opening of the container-shaped concave part 4 is closed tightly by the cover glass 6. In this manner, the aqueous solution 1 containing the protein comes to a supersaturated state due to evaporation of moisture in the course of time, and eventually a crystal 7 of protein is precipitated within the foregoing aqueous solution 1.
After obtaining the protein crystal 7, the crystal 7 is subject to analysis of crystal structure. For performing the crystal structure analysis, a diffraction intensity measurement of the crystal is conducted using an X-ray diffraction measurement apparatus. Therefore, it is required to take the crystal 7, which has grown in the aqueous solution 1, out of the concave part 3 of the well solution retention plate 2. For this purpose, in one of prior arts, first as shown in FIG. 15(a), the protein crystal 7 is picked up from within the concave part 3 along with a part of the aqueous solution 1 with the use of a tool 8 equipped with a nylon ring, which is generally referred to as a nylon loop (of which diameter is approximately 1 mm or less). Normally, this operation is carried out manually under a microscope. As shown in FIG. 15(b), the protein crystal 7 is held along with the aqueous solution 1. Subsequently, although not shown, after moisture of the aqueous solution 1, which is retained within the ring of the tool 8, is substituted by a defrost, the tool 8 holding the protein crystal 7 and the defrost 1′ is mounted into a mounting tube 9. Then, the defrost 1′ is quickly frozen in order to protect the crystal 7 and, thereafter, as shown in FIG. 15(c), the protein crystal 7, which is held within the ring of the tool 8, is irradiated with an X-ray to carry out measurement of a diffraction intensity.
To produce a biopolymer crystal being typically represented by a protein and to conduct an X-ray diffraction measurement of the obtained crystal, the above-mentioned method has been heretofore commonly utilized. In this conventional method, when the protein crystal 7 has grown within the aqueous solution 1, to provide the foregoing crystal 7 for conducting an X-ray crystal structure analysis, it is necessary to take each individual crystal 7 out of the concave part 3 of the well solution retention plate 2, and further mount the crystal 7 into the dedicated mounting tube 9. However, since this operation is normally carried out manually, several problems exist in that in the case of conducting the crystal structure analysis of a great deal of protein crystal, this operation is extremely inefficient and requires a lot of time as well as a larger amount of labor of an operator.
Additionally, in a method utilizing a heterogeneous nucleation in which method a crystal nuclear of a protein is formed and grown on a surface of a solid substrate, the crystal grows in the state of being adsorbed onto the surface of the solid substrate. Accordingly, in the case where the crystal has grown up to be a large size of crystal, because entire lower face of the crystal is adhered to the substrate, a further problem exists in that the crystal is broken and cracked therein at the time of taking out the crystal, eventually making it impossible to subject the crystal to the crystal structure analysis.